Vitamin



Patented Dec. 16, 1941 UNITED STATES PATENT OFFICE VITAMIN No Drawing.Application May 9, 1939, Serial No. 272,699. In Germany June 19, 1935 4Claims.

The present invention, which is a continuation in part of our copendingU. S. application Ser. No. 30,156, filed July 6, 1935, now PatentNo.2,163,659, relates to the class of substances of paramount biologicalinterest commonly designated as vitamins, and more especially to thesubstance, or

group of substances, belonging to this class,

which has hitherto been known under the name of antirachitic vitamin orvitamin D and which is capable of preventing or curing rickets. It hasparticular reference to that modification of vitamin D which isespecially active for chicks.

Our invention relates furthermore to the precursor, the so-calledprovitamin D, from which that vitamin D can be prepared by irradiationwith ultraviolet light.

The invention also includes .the processes of producing thesesubstances.

The history of the research work done in this field of science, aswritten for instance by Waddell and published in July, 1934, in theJournal of Biological Chemistry (vol. 105, p. 711-739) revealsincessant, but on the whole fruitless efforts, on the part of quite anumber of highly skilled investigators, to trace the nature andconstitution of the antirachitic principle especially active for .chicksand eventually to isolate and produce it, if

not on a large scale, at least in the laboratory. Waddell, as far as weare informed, was the last and most successful worker in this field, andhis discovery, that the antirachitic factor of irradiated cholesterol isas potent for chicks as the from ergosterol, which one had hithertobelievedto be the sole precursor of vitamin D, was a great step forwardin this still mostly unknown field ofbiological science. ings may havebeen from 'a scientific point of view, they did not contribute much tothe solution of the problem of how to isolate this active principle orits precursor and, which is still more important, to produce it on alarge scale so as to bring it within reach of every poultry farmer.

All we knew with regard to manufacture of antirachitic activepreparations on an industrial scale was, that they could be produced byirradiation, with ultra-violet rays, of animal and vegetable mattercontaining unsaponifiable lipoids,

However important his find rickets in the same way as the naturalvitamin D in cod liver oil, there exists a marked difference betweenthese two vitamins. It had been found that preparations of these twovitamins possessing the same potenciescompvted by comparison with theinternational standard preparations for vitamin D on rats- -when testedon chicks, did not present the same activity, but that from 30 to timesas many international units of irradiated ergosterol were required toproduce the same antirachitic efiect, as of cod liver oil. Waddell foundthat irradiated commercial choles'terol has about the same activity forchicks as cod liver oil, if compared on the base of the same number ofinternational rat units.

The highest amount of provitamin ever encountered by prior investigatorsin cholesterol (from the egg of a hen fed with ergosterol) was 0.32%,and in those cases known to us, where no ergosterol had been fed to theanimals, not even' this figure has ever been reached. Now, sinceaccording to Waddells paper he never obtained more than 5 internationalunits per milligram irradiated cholesterol and since our owninvestigations had convinced us, that it would be impossible, accordingto the methods hitherto followed, even if starting from a cholesterolpreparation containing as much as 0.3 per cent provitamin, to obtainmore than 30 international units per mg. irradiated cholesterol; sincemor'eoverthe low degree of solubility of cholesterol in oil would notfavor the commercial preparation of solutions possessing more than 200to 300 international units per cubic centimetre, and inview of the highcosts of production we concluded that the cholesterol material hithertoused was not a suitable source of provitamin roduction'and that we wouldhave to search for a other more prolific and easily available rawmaterial.

We have succeeded in tracing not only such a material availableeverywhere and in unlimited quantity, but also the means for recoveringfrom this material on a commercial scalepreparations rich inantirachitic principle especially active on chicks, and we are now ableto not only provide I the market at low cost with high percentageprovitamin D preparations excellently suited. for the production of thesaid antirachitic principle, but

and before Waddells publication the provitamin also to isolate thechicken-provitamin D as a welldefined crystalline compound.

Our invention, therefore, comprises the method of producing preparationsrich in provitamin by starting from materials never before tested forsuch purpose, the new preparations obtained in manner, further the purechicken provitamin D itself and finally the antirachitlcally activepreparations derived therefrom.

We are now going to describe our invention and how the same is to beperformed.

THE STARTING MATERIAL may however also extract the dried material in anordinary extractor, or dry and extract it in a rotating vacuumextractor, however taking care sight appears to be little founded; itoccurred to us that, since ergosterol, although traceable in all plants,is far more preponderant among the sterols of the lower plants(cryptogams) than among those of the higher plants (phanerogams)possibly similar conditions might prevail amongst animals in regard tothe contents of that sterol in which irradiation with ult aviolet raysgenerates the active vitamin principle. .We thought that thisprovltamin-sterol I right be much more preponderant among the sterols ofthe lower animals (Invertebrata) than in the cholesterol among those ofthe higher animals (Vertebrata). Followingthisidea we started aninvestigation of the sterols of the Invertebrata, on which as sources ofthe antirachitically activatable provitamin nothing could be'found inliterature. This investigation was not encouraged by the fact that inmany instances the sterols found in these materials had been proved tobe different from cholesterol, so that it seemed doubtful whether theprovitamin D, which we might ultimately trace therein, might be thechicken-provitamin D we were looking after.

In view of an extended series oftests made with sterols recovered fromvarious kinds of Invertebrata, including Annelida, Arthropoda andMollusca, we feel authorized to say that we have in fact traced thefirst and probably the only way of producing the chicken-provltamin D onthe largest possible scale. For thesterol fraction of most of theanimals of the large family of Invertebrata, which we have tested,

- have been found to yield many times, and some up to and beyond thehundredfold, of the yield of provitamin D normally recoverable from thesterols of the Vertebrata.

Obviously,.since these raw materials are living organisms, theprovltamin content in different individuals of the same species will notbe constant, but is bound to vary according to the stage of development(age), conditions of living, season, climate, etc.

METHOD OF PRODUCING PREPARATIONS RICH IN CHICKEN PROVITAMIN D In thepreparation of unsaponiflable lipoids and sterols from Invertebrata wehad to keep in mind that the provltamin is a rather unstable substance.We prefer starting with whole animals in a state as fresh as possible,which are material is next extracted with light petroleum, 1

until the fat is completely removed, the petroand dried with anhydroussodium sulfate. We

' leum fractions are united, washed with water not to raise thetemperature'above 50 C. or

temperature with a solution of 50 parts by weight KOI-I in 50 partsdistilled water and 500 partsalcohol of 96%, calculated on 100 parts ofthe residue. This mixture is heated two hours on a steam bath under thereflux condenser. The soap solution is cooled and diluted with distilledwater to an alcohol concentration of 15- to 20%. From this solution theunsaponifiable .fraction may for instance'be extracted five times withethyl ether, the ether washed five times with distilled water anddistilled off, care being taken that the contact with ether be as shortas possible.

Alternatively the diluted soap solution may be treated with a solutionof a calcium salt, equiva lent to the amount of KOH used, whereby theunsaponifiable fraction is precipitated along with the lime soaps. TheKOH in excess is converted into K2CO3 by adding CO2 or a NaHCOasolusoluble in methyl alcohol, that can be decanted or filtered off. Inthese cases the yield of sterol fraction is impaired, unless the motherliquors are treated further in a manner adapted to the circumstances,because the impurities tend to keep the sterol in solution. If theunsaponifiable matter should contain a high percentage of coloringmatter such as for instance carotinoids, special care should be taken toavoid losses of provltamin by the destructive influence of light and theoxygen of the air.

We have tried to isolate the sterol fraction by precipitation withdigitonin and extracting the precipitate with boiling xylene in theusual way, but this process is unsuitable because the provltamin iseasily destroyed. For the purification of the sterol fraction, which maybe effected for instance by recrystallisation or by distillation in ahigh vacuum, no treatment with adsorbents, such-as charcoal, should beresorted to, because this also leads to destruction of the provltamin.

After the sterol fraction has been purified sufficiently, the absorptionspectrum is measured in the usual way; the absorption spectrum iscompared with that of ergosterol, and the amount of ergosterol, thatwould give the same absorption, is calculated in per cents of the amountof sterol used. This percentage is used to indicate the amount ofprovltamin present.

As will be seen from the foregoing description, we are operating withthe fresh raw material, taking care to avoid heating and quiteespecially boiling steam.

ACTIVATION" OF THE CHICKEN-PROVITA- MIN D PREPARATIONS BY IRRADIATIONwith water and drying with oxygen under continual stirring of thesolution with light from which the wave lengths below 284' millimicronshave been filtered off by a xylene filter, and stopping the irradiationbefore more than about 50% of the provitamin has been transformed, asdescribed-in our paper in Biochemical Journal 25, 1001-1002 (1931). Forthe present experiments we used an all-quartz absorption cell connectedwith the irradiation apparatus, and we dissolved the different sterolfractions in ether free of peroxide in such a concentration that theabsorption was practically the same in every case. We chose theconcentration so small that practically the whole energy of the mercuryline 313 millimlcrons and themain part of the line 302 millimicrons werenot absorbed in the irradiation apparatus.

After irradiation the absorption spectrum of the irradiated solution canbe measured again.

We found that the photochemical reaction as represented by changes inthe absorption spectrum is the same as in the case of ergosterol (seeour paper in Bio-chem. Journ. 23, 1929, Therefore the absorptionspectrum after irradiation enabled us to calculate the degree oftransformation in the same way as with ergoster'ol; and the rat testshave confirmed this in every respect.

Irradiation being completed, the solution was poured intoantirachitically inactive arachis oil in vacuo and the ether wasdistilled off. The oil solution was then diluted to the properconcentration for the rat and chicken tests.

THE CHICKEN TEST In the chicken test we started with one-day oldchickens which were fed during 34 weeks on a ration consisting of 12%casein, ground whole wheat, 59% ground whole yellow maize, 1% driedyeast, 2% CaCOa and 1% NaCl, 1 arachis oil containing the vitamin Dpreparation being added to the mixture.

We found that 70 international units of codliver oil vitamin D per 100grams ration was enough to guarantee a normal growth and boneformationof the chickens up to 3 or 4 weeks. A smaller amount of vitamin D led toan abnormal bone-formation, which could be detected radiographically. Wehave thus been able to perform a quantitative estimation of vitamin Dwith chickens as test animals, which need not be gone into in detail.

We will only give a representative example: 181

mgs. of a preparation of sterol from mussels,

containing, according to the spectrum, 12 per under continuous stirringin vacuo with the light,

filtered through a xylene solution, of a mercury vapor quartz lamp,until about to per cent of the provitamin present in the substance wastransformed. The irradiated solution, while stillin the vacuum, was thenentered into 190 cubic.

ing material yielded about'140,000 international units of vitamin D,which is about 800 international units per milligram.

EXAMPLES OF THE PRODUCTION OF HIGH PERCENTAGE PREPARATIONS OF CHICK- ENPROVITAMIN D In thepractical operation of the process of producingpreparations rich in provitamin we proceeded for instance as follows:

EXAMPLE 1 M ollusca filtration the ether was distilled off and the gramsfatty matter, which remained over, were saponified by heating themduring two hours under the reflux condenser with a solution of 20 gramsKOH in 20 cubic centimetreswater andv 200 cubic centimetres alcohol of96%. After cooling, the soap solution was poured, into 1 litre distilledwater and the aqueous soap solution centimetres arachis oil and theether evaporated in vacuo. The antirachitic effect of the oil solutionwas tested on, rats in comparison to the international standard solution'of vitamin D and further .on chicks in comparison with a cod-liver oilcontaining 100 international units (rat units) vitamin D per cubiccentimetre. The rat test showed the oil solution to contain about 750international units per cubic centimetre. The chick test showed theoil-solution to be about 7 /2 times stronger than thecod-liver oil. Thus.181

milligrams sterol from mussels forming the startshaken five times withethyl ether, 1.1 litres of which were required altogether. The ethericsolution was now shaken fiveutimes with distilled water, 100 cubiccentimetres water being used each time, whereupon it was dried withNa2SO4 and filtered. On removal of the ether by distillation thereremained over a solid reddish yellow mass weighing about .7.5 grams.This crude unsaponifiable fraction of the fatty matter was nowrecrystallized from 50 cubic centimetres alcohol.

of 96%, once more from 25 cubic centimetres of such alcohol and finallyfrom cubic centimetres methyl alcohol.

'I-he sterol fraction recovered in this manner weighed 2.5 grams andproved to be a light yellow crystalline mass. By measuringthe-absorption spectrum of these crystals wefound the characteristicabsorption bands of the provitamin to be present in a strengthindicating a provitamin content of the substance of 16 per cent. Asmentioned above, we took care to keep the treatment with ether as shortas possible. content of 16% of the sterol fraction from mussels, asdescribed in this example, corresponds after irradiation to anantirachitic activity of about 1600 international units per milligram.

EXAMPLE 2 '2.2 kilograms periwinkle (Littorina littorca) were crushedtogether with their shells in a mortar and then comminuted in a meatchopper, whereupon the mass was treated with 2 litres alcohol. Afterdilution with water the alcohol was shaken with petroleum ether and thedried mass also extracted with petroleum ether. After washing, dryingand evaporating the ether, we obtained a residual fatty matter weighing11.8 grams, which was now saponified by heating it 2 hours under thereflux condenser with a solu tion of 6 grams KOI-I in 6 grams water and50 cubic centimetres alcohol of 96%. The soap solution was diluted with250-"cubic centimetres distilledwater and the unsaponifiable fractionseparated by means of ethyl ether. tained 1.85 grams unsaponifiablematter from which we produced by recrystallization from al-.

The provitamin We thus ob-' cohol and methyl alcohol the sterol fractionunder the form of light yellow crystals weighing 0.49 gram.

The absorption spectrum showed these crystals to contain 26 per centprovitamin D corresponding after irradiation to an activity of about2,600 international units per milligram.

EXAMPLE 3 Annelida The alcoholic extracts which on being recrystallizedfrom alcohol, and

methyl alcohol yielded a sterol fraction of white crystals weighing 0.3gram. The preparation was found to contain 11 per centprovitamincorresponding after irradiation to an antirachitic activity of about1,100 international units.

EXAMPLE 4 7 800 grams small fresh water-worms (Tubifezc) were comminutedin a meat chopper and treated with alcohol 01' 96% and petroleum etherof boiling point 40-60 C. in the way described in Example 2. Allpetroleum ether extracts were combined, washed with water, dried overNazSOr and evaporated. The extract weighing 13.0 grams was saponifiedand the unsaponiiiable part was extracted with ethyl ether. We obtained2.5 grams which were recrystallised from alcohol and methyl alcohol,yielding a white crystalline sterol fraction weighing 1.12 grams. Thesecrystals were found to contain 17 per cent provitamin corresponding,after irradiation, to an antirachitic activity of about 1,700international units per milligram.

As compared with the maximum of 5 intemational units per milligram ofirradiated cholesterol obtained by Waddell and the fraction of one percent provitamin content of any cholesterol preparationhitherto obtainedfrom vertebrate animals, the sterol fraction of the invertebrate animalstested according to the above examples showed the chicken provitaminD'contents to In the production of this pure provitamin D we convertedpart of the sterol fraction recovered in accordance with Example 1 intothe acetate by treating same with acetic anhydride and pyridine. Themixture of acetates thus obtained was dissolved in a mixture of benzeneand light petroleum and adsorbed in a column of alumina preparedaccording to Brockmann. Bylixiviation with the same solvent a largeportion of the acetates of the inactivatable sterols could be removedand the adsorbed material was finally recovered by lixiviation withbenzene, to which some methyl alcohol was admixed. In this way wesucceeded in concentrating the provitamin acetate about two and a halftimes in a single adsorption operation. Starting from an acetate mixturecontainingabout 7 per cent provitamin acetate, we obtained, after 3adsorption opera tions. a mixture containing about 60 per centprovitamin acetate. This mixture was recrystallized three times frommethyl alcohol and yieldedan acetate, the absorption spectrum of whichcorresponded to the spectrum of 94% by weight of ergosterol acetate.Three further recrystallizations did not materially alter thisabsorption spectrum; the melting point changed only slight- 1y.

We consider this acetate to be the pure provitamin acetate melting at160 to 161 C. (uncorrected) and having a [11:11): 78 (dissolved inbenzene). For the sake of comparison we note the melting point of pureergosterol-acetate as 175.3 to 176 C. (uncorrected) and [1110: 96.

This shows that the provitamin from the sterol here used as startingmaterial is different from ergosterol.

The provitamin itself was obtained by saponification of the acetate.After many recrystallizavary between 2.5 and 26 per cent, being in anyEN-PROVITAMIN D We have further succeeded in concentrating the sterolsobtainable from the fatty matter in invertebrate animals to the extentof enabling -us to produce a pure crystallized provitamin melting at149.5 to 150 0., whose specific rotation, measured in benzene, is [a]D=-118.

\ PREPARATION" OF CHRYSTALLIZED CHICK- tions from methyl alcohol itsmelting point was found at 149.5 to C. in contradis'tinction toergosterol melting at 163.5 to 164 C. The specific rotation of thechicken-provitamin D in benzene [aim was found to be -118, as against-136 for ergosterol.

The absorption spectrum of the pure provitamin differs only slightlyfrom that of ergosterol.

This pure provitamin is further differentiated from ergosterol by thefact that, whereas the active principle formed in ergosterol(calciferol) has an activity. of about 1,000 international chick unitsper milligram, the active principle formed in said provitamin has anactivity ranging from about 10,000 to 40,000 international chick unitsper milligram.

This pure provitamin is a typical sterol, which is precipitated bydigitonin in alcoholic solution. It is decomposed relatively rapidly inetheric solution and in boiling acetone, if exposed to the air. Inshort, it behaves rather like ergosterol,

but it seems to be a little more susceptible to oxidation.

We have furthermore succeeded in producing crystallized chickenprovitamin D from the sterol fraction recovered in accordance withExample 3, by converting it into the isobutyrate by treating withisobutyric anhydride and pyridine and recrystallizing 'theresultingisobutyrate from alcohol. The resulting mixture'of isobutyrates (10.15gr. containing Il of provitamin isobutyrate) was dissolved in a mixtureof benzene and. light petroleum and repeatedly absorbed in a column ofalumina prepared according to Brockmann, followed by lixiviation by thesame solvent. In this way a satisfactory separation between theisobutyrates of the inactivatable acetic anhydride during thirty minutesand recolumn.

fraction recovered in accordance with Example 15 4, by converting itinto the acetate by treating with acetic anhydride and pyridine, and theresulting acetate recrystallized from acetic anhydricle and methylalcohol-ether. 12.3 g. acetate containing-26.4% of provitamin acetatewere dissolved in a mixture of benzene and light petroleum and in tworuns adsorbed in a column 'of alumina prepared according to Brockmann.By

lixiviation with the same solvent a large portion of the acetates of theinactivatable sterols could be removed with the first 700 ccm. of liquidrunning through. The next two litres of solvent running throughcontained (in toto for the two runs) 5.1 g. with 47% ofprovitamin-acetate. This material was again treated in the same way andyielded 1,110 mg. of a material-which was spectroscopically pureprovitamin-acetate. After three crystallizations from methyl alcohol thepreparation (672 mg.) melted at 1505-1545 C. and had a rotation angle of[1111): '8'7 (in benzene) After saponification and recrystallisaacetatewas obtained. After three crystallisations from methyl alcohol thepreparation melted at 135-136 C. and the rotation angle in benzene was[u]o=85.5.' After saponification and recrystallisation frommethyl-alcohol a chickenprovitamin sterol'was obtained in the form ofwhite crystals melting at 137-137.5 C. and having a specific rotation inbenzene of [a]D=-124. Various changes may be made in the detailsdisclosed in the foregoing specification without departing from theinvention or sacrificing the advantages thereof. v

The term international chick uni as used in the claims is to beunderstood to mean the same anti-rachitic activity on chicks as would betion from methyl alcohol a crystalline chicken- ,provitamin-sterol wasobtained. in the form of white crystals melting at l43-146 C. and havinga specific rotation in benzene of [a]D= -127.

We have furthermore succeeded in producing crystallised chickenprovitamin D from the sterol ganic solvent, submitting the solution ofesters to a fractional adsorption in an adsorption column containingalumina, and separating the fraction containing the main part of theprovitamin ester.

2. A process for producing provitamin preparations from a crude extractof an invertebrate animal material,.comprising the steps of treating thecrude extract to esterify the sterol content thereof to a saturatedfatty acid ester, dissolving said ester in a non-polar organic solvent,sub-- mitting the solution of the ester to a fractional adsorption in anadsorption column containing tions from a crude ;extract of aninvertebrate fraction recovered in accordance with Example 2,

by converting it into the acetate by boiling with crystallizing theresulting acetate from boiling ethyl alcohol. This mixture of 25 'g.periwinklesterol-acetates' containing 6.6% of provitamin- I acetate wasrepeatedly treated in an adsorption same solvent. The liquid which runsthrough first, contains a large part of the acetates .of theinactivatable sterols with a small amount of provitamin-acetate. T ispart is collected sepa-' rately and lixiviation is carried on with thesame solvent until practically all the adsorbed provitamin acetate hasbeen dissolved from the The residue obtained after evaporating in vacuothe solvent from the first collected filtrate is combined withcorresponding residues from other runs and subjected to the ametreatment again. This treatment was repeated with animal material,comprising the steps of treating the crude extract to esterify thesterol content thereof to an acetic acid ester, dissolving the ester ina non-polar organic solvent, submitting the solution of the ester to afractional adsorption in an adsorption column containing alumina, andseparating the fraction containing the main part of the provitaminester.

4. A process for producing provitamin preparations from a crude extractof an invertebrate animal material, comprising the steps of treating thecrude extract to esterify the sterol content thereof to an isobutyricacid ester, dissolving the ester in a non-polar organic solvent,submitting the solution of the ester to a fractional adsorption in anadsorption column containing alumina, and separating the fractioncontaining the main part of the provitamin ester. 1

- C. BOER-v. n. WURFT', Administratria: of Estate of Albert GerhardusBoer, Deceased;

- JOHANNES VANZNEKEZRK.

ENGBERT HARMEN REERINK. .AAR'I rm WIJK.

of 731 mg. of a spectroscopically pure provitamin

